Anti-tumor activity of Ea-4-peptide of pro-IGF-I

ABSTRACT

Compositions of pro-IFG-I E-peptides for the treatment and amelioration of tumor-producing diseases, and methods for their utilization.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a divisional application claiming priority under 35 U.S.C. §§120 and 121 to U.S. patent application Ser. No. 10/647,732, filed Aug.25, 2003, now U.S. Pat. No. 7,250,169; which is a continuation of U.S.patent application Ser. No. 09/669,642, filed Sep. 26, 2000, now U.S.Pat. No. 6,610,302; which is a continuation-in-part of U.S. patentapplication Ser. No. 09/120,818, filed Jul. 22, 1998, now U.S. Pat. No.6,358,916, the disclosures of which are hereby incorporated by referencein their entirety.

INCORPORATION BY REFERENCE

The present application hereby incorporates by reference, in itsentirety, the Sequence Listing, and identical CRF of the SequenceListing filed previously with the United States Patent and TrademarkOffice in association with U.S. patent application Ser. No. 10/647,732(US Patent Publication #: US 2005-0250687 A1); filed: Aug. 25, 2003;entitled: Anti-Tumor Activity of Ea-4-Peptide of Pro-IGF-I. Theinformation contained in the Sequence Listing of the instant applicationis identical to the sequence information contained in the computerreadable form and contains no new matter.

FIELD OF THE INVENTION

The present invention generally relates to anti-tumor peptides. Inparticular, the present invention relates to use of specified pro-IGF-1E-peptides in the treatment and amelioration of tumor-producingdiseases. Certain pro-IGF-1 E-peptides are shown to decrease tumorgrowth and to reduce metastasis.

BACKGROUND

Attention recently has been focused on the biological activities of theproteolytically-processed polypeptides from post-translational modifiedpeptide hormones. For example, the C-peptide of pro-insulin has longbeen regarded to be biologically inactive except for a possible role inthe folding of the insulin molecule during its post-translationalmodification. However, Ito et al. (14) have found recently that theC-peptide of pro-insulin is important in restoring vascular and neuraldysfunction and Na+/K+-dependent ATPase activity in diabetic rats.Although a synthetic peptide amide of human b-type IGF-I E-peptide hasbeen shown to exert mitogenic activity by Siefried et al. (5), thebiological activity of the native human E-peptides have not beenidentified.

Insulin-like growth factor-I (IGF-1) is a member of the insulin genefamily and plays an essential role in growth, differentiation,regeneration and metabolism in all vertebrates (2). In all animalsstudied to-date, the primary translation product of IGF-I mRNA containsdistinct regions or domains, including an N-terminal pre-peptide leader,followed by the mature peptide with B, C, A and D domains, and aC-terminus E domain. Due to alternative RNA splicing, two differentforms, the a- and b-type, of IGF-I pro-hormones exist in mammals,differing only in the E-peptide region. In human, mouse and rat, thefirst 15 amino acid residues in the N-termini of the E-peptides ofpro-IGF-Ia and -Ib share identical sequences, and this region isreferred to as the common region. The sub-types of pro-IGF-I's aredistinguished from one another by the amino acid sequences after thefirst 15 common amino acids, known as the variable regions of theE-peptides (3).

In mammals, the signal peptide and the E-peptide are proteolyticallycleaved after translation from the pre-pro-hormone to form a maturepeptide of 70 amino acid residues. The E-peptides of mammalianpro-insulin-like growth factor-I (pro-IGF-I) have long been regarded asbiologically inactive.

Surprisingly, the present inventors have recently discovered thatcertain recombinant rainbow trout pro-IGF-I E-peptides (Ea-2-, Ea-3- andEa-4), like hIGF-I, exhibit a dose-related mitogenic activity in severalmammalian cell lines (Tian et al. (1)).

Four different forms of a-type IGF-I mRNA have been observed in rainbowtrout (rt), Oncorhynchus mykiss, and are designated as IGF-I Ea-1, Ea-2,Ea-3 and Ea-4 (4). Nucleotide sequence comparison of the four size formsof IGF-I mRNAs reveal that the size differences among these mRNA speciesare due to insertions or deletions in the E domain regions of themolecules. The first 15 predicted amino acid residues of the fourE-peptides share identical sequences among themselves as well as withpro-IGF-I E-peptides of human, mouse, and rat. The presence of theC-terminal 20 amino acid residues, sharing 70% identity with their humancounterparts, identifies them to the a-type E-peptides.

The Ea-1-peptide of the rt-pro-IGF-I is a polypeptide of 35 amino acidresidues, comprising the first 15 and the last 20 amino acid residues.Ea-2 and Ea-3 peptides differ from Ea-1 by virtue of either a 12- or27-amino acid residue insertion between the first and last segments ofthe Ea-1-peptide sequence, respectively, while Ea-4 contains bothinsertions. The predicted numbers of amino acid residues in eachE-peptide are, thus, 35, 47, 62 and 74, respectively. There has not beenany report on the presence of b-type IGF-I mRNA in rainbow trout.

While the biological functions of the mature IGF-I peptide have beenintensively studied, the functions of E-peptides have been overlookedand remain to be identified.

Siegfried et al. (5) have recently shown that the amide of a 22-aminoacid residue encoded within the variable region of the E-peptide of thehuman IGF-Ib (IBE₁) exerts mitogenic activity in normal and malignantbronchial epithelial cells. Surprisingly, as set forth above, thepresent inventors have recently found that certain E-peptides from fishpossess mitogenic activity with respect to mammalian cells (Tian et al.(1) report that recombinant rainbow trout Ea-2-, Ea-3- and Ea-4-peptidepossess mitogenic activity in several normal and oncogenic transformedmammalian cell lines including NIH 3T3 cells, human embryonic kidneycells transformed by retrovirus (293GP), human mammary gland tumor cells(MCF-7) and caprine mammary epithelium cells (CMEC) (6)).

Based on the surprising inter-class activity of iethyofauna Pro-IGF-IE-peptides on mammalian cells, and given that trout Ea-2, Ea-3 and Ea-4peptides containing a signal motif for peptidyl C-terminal amidation(4,7) and a bipartite consensus nuclear localization sequence (4,8), thepresent inventors hypothesized that these peptides might possess othernovel biological activities on mammalian cells. In its screening fornovel biological activities of trout pro-IGF-1 E-peptides, the presentinventors undertook several studies of the effects of such E-peptides onmorphology, colony formation activity on a soft agar medium, andinvasiveness of human and trout cancer cells. Surprising and unexpectedanti-tumor activity was discovered.

SUMMARY

The present provides a method for reducing tumor growth and theinvasiveness of cancerous cells in mammals and fish by administration ofa Pro-IGF-I E-peptide obtained from fish.

The present inventors have shown that the treatment of established humanand fish cancer cell lines (MCF-7; HT-29; HepG2, ZR-75-1, SK-N-F1 andHC) and an oncogenic transformed human cell line (293GP) withrecombinant trout Ea-2- or Ea-4-, but not Ea-3-peptide, resulted in adose-dependent induction of morphological change and enhanced cellattachment. They have also shown that the in vitro colony formationactivity of the oncogenic transformed cell line or established tumorcell lines is greatly reduced or diminished by treatment with the troutEa-4-peptide. Furthermore, the invasive activity of HT1080, a knowninvasive cancer cell line, is also reduced many fold by treatment withthe Ea-4-peptide. These results suggest that the Ea-4- and Ea-2-peptidesof rainbow trout pro-IGF-I are able to control the malignant propertiesof cancer cells. The E-peptide-induced morphological changes aresensitive to treatment with α-amanitin or cycloheximide, knowninhibitors of RNA and protein synthesis.

In one embodiment of the present invention there is disclosed a methodof inhibiting proliferation of malignant cells, comprising administeringto the malignant cell at least one E-domain peptide agent. The E-domainpeptide agent is preferably selected from the group consisting of: atrout E-domain peptide, an E-domain peptide homolog, and an E-domainpeptide fusion protein, and may more preferably be an E-domain peptideof a rainbow trout. In a preferred embodiment, trout E-domain peptide isselected from the group consisting of: Ea-2 domain peptide (SEQ ID NO:2)and Ea-4 domain peptide (SEQ ID NO: 4). The E-domain peptide agent maybe administered in a pharmaceutical composition.

In another embodiment of the present invention, there is disclosedmethod of inhibiting the proliferation of malignant cells, comprisingadministering to the malignant cells a nucleic acid encoding an E-domainpeptide agent. Preferably the E-domain peptide agent is selected fromthe group consisting of: a trout E-domain peptide, an E-domain peptidehomolog, and an E-domain peptide fusion protein and more preferably theE-domain peptide is an E-domain peptide of a rainbow trout, inparticular having a sequence selected from the group consisting of: Ea-2domain peptide (SEQ ID NO:2) and Ea-4 domain peptide (SEQ ID NO: 4). Thenucleic acid encoding the E-domain peptide may be administered in apharmaceutical composition.

In yet another embodiment of the present invention, there is disclosed amethod for reducing the invasiveness of malignant cells, comprisingadministering to the malignant cells an E-domain peptide. In suchembodiment, the E-domain peptide agent is preferably selected from thegroup consisting of: a trout E-domain peptide, an E-domain peptidehomolog, and an E-domain peptide fusion protein, and more preferably isan E-domain peptide of a rainbow trout. Preferably, the trout E-domainpeptide is selected from the group consisting of: Ea-2 domain peptide(SEQ ID NO:2) and Ea-4 domain peptide (SEQ ID NO: 4). The E-domainpeptide agent may be administered in a pharmaceutical composition.

And yet in another embodiment of the present invention, there isdisclosed a method for reducing the invasiveness of malignant cells,comprising administering to the malignant cells a nucleic acid encodingan E-domain peptide agent. Preferably, the E-domain peptide agent isselected from the group consisting of: a trout E-domain peptide, anE-domain peptide homolog, and an E-domain peptide fusion protein, andmore preferably is an E-domain peptide of a rainbow trout. Preferably,the trout E-domain peptide—is selected from the group consisting of:Ea-2 domain peptide (SEQ ID NO:2), and Ea-4 domain peptide (SEQ ID NO:4). The nucleic acid encoding the E-domain peptide may be administeredin a pharmaceutical composition.

The above-discussed and other features and advantages of the presentinvention will be appreciated and understood by those skilled in the artfrom the following detailed description and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photomicrograph of human tumor cell lines [293GP, MCF-7,HT-29, HepG2, ZR-75-1, SK-N-F1 (human neuroblastoma cells) andPeoceliposis hepatoma cells (HC)] with and without treatment with Ea-4peptide of trout pro-IGF-I or human IGF-I.

FIG. 2 is a photomicrograph of oncogenic transformed/established tumorcell lines 293GP and MCF-7 cells cultured under control conditionsalone, control conditions+α-aminitin, an RNA synthesis inhibitor, orcontrol conditions+cycloheximide, a protein synthesis inhibitor.

FIG. 3 is a photomicrograph of oncogenic transformed/established tumorcell lines 293GP and ZR-75-1 cells cultured in basal medium fortwenty-four hours and then treated with Ea-2-, Ea-3, or Ea-4-peptide.

FIG. 4 is a photomicrograph of oncogenic transformed/established tumorcell lines 293GP, MCF-7 or HT-29 plated on basal medium containing 0.4%agar without fetal bovine serum and various concentrations of h-IGF-I ortrout Ea-4-peptide.

FIG. 5 a is an illustration of a procedure undertaken to transfect agene construct containing a green fluorescence gene (EGFP) or Ea-4peptide gene and green fluorescence gene (EGFP) into MDA-MB-231 breasttumor cells, and a method for determining colony formation activity ofthe transfected Ea-4 positive clones from EGFP controls.

FIG. 5 b are photomicrographs of MDA-MB-231 breast tumor cellstransfected with EGFP alone, and EGFP and Ea-4, with a graph interposedthere between that indicates the relative colony formation under eachscenario.

FIG. 6 is a graph illustrating the effect of Ea-4-peptide on tumorgrowth, induced by injection of ZR75-1 cells in nude mice.

FIG. 7 is a table illustrating the effect of Ea-4-peptide on theinvasion activity of HT-1080 cells on Matrigell.

DETAILED DESCRIPTION

The present invention provides a method and compositions useful forreducing tumor growth and tumor cell invasiveness.

Novel biological activities are shown to be associated with E-peptidesof the rainbow trout pro-IGF-I. In addition to mitogenic activity, troutpro-IGF-I Ea-2 and Ea-4-peptide possess activities including inductionof morphological changes, enhancing cell attachment, restoringanchorage-dependent cell division behavior, eliminating anchorageindependent cell division, and reducing the invasiveness of aggressivecancer cells. Further, in vivo studies conducted in the presentinventor's laboratory demonstrates that tumors developed from theEa-4-peptide treated ZR-75-1 cells in nude mice are substantiallysmaller than those developed from untreated cells. Since similarmorphological changes have also been induced in a hepatoma cell line ofPeoceliposis lucida (desert guppy) by treatment with the troutEa-4-peptide, this rules out the possibility that the effect of troutpro-IGF-I E-peptides observed in human cancer cells is the consequenceof artifact.

The invention takes advantage of the biological activities of E domainpeptides. In certain embodiments of the methods of the invention, Edomain peptides of a trout are used. As used herein, the term “Epeptide” or “E domain peptide” refers to a peptide that forms an Edomain of IGF-I.

Peptides which are at least about 70% identical, preferably at leastabout 80% identical, and more preferably at least about 90% identical tothe trout E domain peptides described herein can also be used, providedthat the peptides have at least one of the biological activities of an Edomain peptide, as described herein. Such peptides are referred toherein as “E domain peptide homologs”.

The E domain peptide can also be part of a fusion protein comprising theamino acid sequence of an E domain peptide or an E domain peptidehomolog, fused to an additional component. Additional components, suchas radioisotopes and antigenic tags, can be selected to assist in theisolation or purification of the peptide or to extend the half life ofthe peptide; for example, a hexahistidine tag permits ready purificationby nickel chromatography. A fusion protein comprising an E domainpeptide is referred to herein as an “E domain peptide fusion protein”.

The trout E domain peptides, E domain peptide homologs, and E domainpeptide fusion proteins described herein are referred to collectively as“E domain peptide agents.” In the methods of the invention, at least oneE domain peptide agent is used; more than one E domain peptide agent canalso be used. If more than one E domain peptide agent is used, theagents can be different types. For example, an E domain peptide and an Edomain peptide homolog can be used concurrently, as can an E domainpeptide homolog and an E domain peptide fusion protein. Alternatively orin addition, if more than one E domain peptide agent is used, the agentscan be of the same type. For example, two E domain peptides, such as theEa-2 domain peptide and the Ea-4 domain peptide, can be usedconcurrently.

EXAMPLES Cell Cultures Utilized in Studies

For routine maintenance of cell cultures, MCF-7 cells (ATCC, humanbreast cancer cells) and ZR75-1 cells (ATCC, human breast cancer cells)were cultured in F12/DMEM supplemented with 10% fetal bovine serum(FBS), and 10 ng/ml of insulin (Gibco-BRL, Rockville, Md.), HT-29 cells(ATCC, human colon cancer cells) in F12/DMEM (GIBCO/BRL) with 10% FBS,HepG2 cells (ATCC, human hepatoma cells) and 293GP cells (transformedhuman embryonic kidney cells provided by Dr. J. C. Burns at UCSD) inDMEM with high glucose, HC cells (Peoceliposis lucida hepatoma cells,provided by Dr. Larry Hightower at the University of Connecticut,Storrs, Conn., U.S.A.) in CO₂-independent medium and SK-N-F1 cells(ATCC, human neuroblastoma cells). Cells were incubated at 37° C. undera humidified atmosphere of 5% or 10% CO₂. All tissue culture media usedin this study were purchased from Gibco-BRL (Rockville, Md.).

Example 1 Morphological Changes Induced by Ea-4-Peptide

Overview: To determine whether the Ea4-peptide may possess biologicalactivities other than the mitogenic activity reported by Tian et al (1),oncogenic transformed or established cancer cells derived from human andfish (293GP, MCF-7, HT-29, HepG2, ZR-75-1, SK-N-F1, or HC cells) werechallenged with the peptide and gross morphological changes recorded.

Methodology: Approximately 1-2×10⁵ of MCF-7, HT-29, HepG2, 293GP, HC orSK-N-F1 cells re-suspended in their respective basal medium withoutfetal bovine serum (SF), were plated in a 6-well culture chamber. Priorto plating cells, an acid-washed coverslip was placed in each well ofthe culture chamber. Various amounts of the trout Ea-4-peptide (2.1 nM)or human IGF-I (hIGF-I, 2.5 nM) were added to each well and the cellcultures were incubated at 37° C. under a humidified atmosphere of 5%CO₂. Coverslips were removed from the culture chamber 24 h afterinitiation of the treatment and observed under an Olympic invertedmicroscope equipped with differential interference phase contrast (DIC)objective lenses (final magnification, 200×).

Results: FIG. 1 is a photomicrograph of human tumor cell lines [293GP,MCF-7, HT-29, HepG2, ZR-75-1, SK-N-F1 (human neuroblastoma cells) andPeoceliposis hepatoma cells (HC)] with and without treatment with Ea-4peptide of trout pro-IGF-I or human IGF-I. As can be seen in FIG. 1,individual cells in the serum-free medium, with or without h-IGF-Itreatment, exhibited rounded morphology and were loosely attached to theculture dish. Cells, however, treated with the Ea-4-peptide wereflattened out and attached tightly to the culture dish. Furthermore, theEa-4-peptide treated cells developed several pseudopodia-like structures(multiple processes) to establish contact with neighboring cells likethat seen in non-transformed or non-malignant cells. After several daysof culturing in the serum-free medium, cancer cells treated with orwithout hlGF-I grew into colonies, whereas those treated withEa-4-peptide spread out in the culture dishes and grew into monolayers(data not shown). The latter are common characteristics exhibited byuntransformed or non-malignant cells.

Example 2 Effect of Inhibition of Synthesis of RNA or Protein onMorphological Changes Induced by Trout Ea-4-Peptide

Overview: The morphological changes induced by the Ea-4-peptide could bethe result of direct interaction of the Ea-4-peptide with pre-existingproteins in the cell or with newly synthesized proteins induced by theEa-4-peptide. To determine whether the morphological changes induced byEa-4 peptide required synthesis of new proteins or RNA, tumorgeniccell-lines were treated with the peptide in and outside of the presenceof known RNA synthesis inhibitors and protein synthesis inhibitors.

Methodology: 293GP and MCF-7 cells were cultured under the conditionsdescribed in above and then treated with 2.1 nM of the troutEa-4-peptide. The cells were then treated with α-amanitin (10 μg/ml), anRNA synthesis inhibitor, or cycloheximide (1 μg/ml), a protein synthesisinhibitor. Twenty fours after initiation of treatment with theEa-4-peptide and the inhibitors, cells are observed under an Olympicinverted microscope equipped with differential interference phasecontrast (DIC) objective lenses (200× magnification).

Results: FIG. 2 is a photomicrograph of oncogenictransformed/established tumor cell lines 293GP and MCF-7 cells culturedunder control conditions alone, control conditions+α-aminitin, an RNAsynthesis inhibitor, or control conditions+cycloheximide, a proteinsynthesis inhibitor. As shown in FIG. 2, the morphological changes inthe 293GP and MCF-7 cells, induced by the Ea-4-peptide, were abolishedby treatment with cycloheximide or .alpha.-aminitin. The viability ofthe inhibitor-treated cells was determined by the dye extrusion assayand the results showed that inhibitor-treated cells remain viable (datanot shown). These results suggest that the morphological changes inducedby the Ea-4-peptide may result from expression of genes that areinactivated during oncogenic transformation or tumor development sincethese experiments were conducted with oncogenic transformed orestablished tumor cells. The syntheses of new RNA and proteins appear tobe required for the Ea-4-peptide induced morphological changes.

Example 3 Effect of Ea-2, Ea-3 and Ea-4 Peptides on Induction ofMorphological Changes in ZR-75-1 and 293GP Cells

Overview: A study was undertaken to determine if there was adifferential effect of three of the known Ea-4-peptides of the rainbowtrout on the induction of morphological changes.

Methodology: ZR-75-1 and 293GP cells were cultured in their respectivebasal medium following conditions described above. Twenty four hoursafter treatment with various concentrations of Ea-2 (SEQ ID NO:2), Ea-3(SEQ ID NO:3) or Ea-4 (SEQ ID NO: 4)-peptide, cells were observed underan Olympic inverted microscope equipped with differential interferencephase contrast (DIC) objective lenses (200× magnification).

Results: FIG. 3 is a photomicrograph of oncogenictransformed/established tumor cell lines 293GP and ZR-75-1 cellscultured in basal medium for twenty-four hours and then treated withEa-2-, Ea-3, or Ea-4-peptide. Although both Ea-2- and Ea-4-peptides areable to induce morphological changes in 293GP or ZR-75-1 cells, the Ea-3peptide fails to induce any visible morphological change under theidentical culture conditions (FIG. 4). This observation suggests thatthe domain of the E-peptide responsible for the induction ofmorphological changes in the 293GP or ZR-75-1 is not present in theEa-3-peptide (1).

Example 4 Effect of Ea-4-Peptide on Colony Formation Activity

Overview: An obvious change in the characteristics of normal cells afteroncogenic transformation is the lost of contact inhibition andanchorage-dependent cell division behavior (10). This behavioral changein oncogenic transformed or cancer cells can be easily demonstrated invitro by the colony formation assay in a semi-solid medium (11). Sincetreatment of oncogenic transformed cells or cancer cells with theEa-4-peptide results in morphological change and increased cellattachment, it is conceivable that this protein may also affect theanchorage-independent cell division behavior of oncogenic transformed orcancer cells. To test this hypothesis, a colony formation assay wasconducted following the methodology described by Yang (9).

Methodology: About 2×10⁴ of MCF-7, HT-29 or 293GP cells at log phasewere plated in their respective basal medium without FBS but containing0.4% purified agar (Difco laboratories) and supplemented with variousconcentrations of hMGF-I (2.6 nM or 5.2 nM) or trout Ea-4-peptide (2.1nM or 8.4 nM), in six-well culture chambers. After the medium wassolidified, each well was overlaid with 1 ml of their respective mediumsupplemented with same concentration of hMGF-I or trout Ea-4-peptide.The plates were incubated at 37° C. in a humidified incubator with 5%CO₂ and examined daily under an inverted microscope for 2 weeks.Colonies were observed under an Olympic inverted microscope equippedwith phase contrast objective lenses (40× magnification). The viabilityof cells at the conclusion of the experiment was confirmed by the dyeextrusion assay with trypan blue.

Results: FIG. 4. is a photomicrograph of oncogenictransformed/established tumor cell lines 293GP, MCF-7 or HT-29 plated onbasal medium containing 0.4% agar without fetal bovine serum and variousconcentrations of h-IGF-I or trout Ea-4-peptide. As shown in FIG. 4,visible colonies were developed from three transformed cell lines grownin a serum-free soft agar medium with or without hMGF-I treatment, butno colony developed from any of the three cell lines cultured in theserum-free medium supplemented with recombinant Ea-4-peptide. Theseresults show that the Ea-4-peptide is able to restore theanchorage-dependent cell division in oncogenic transformed or tumorcells. Furthermore, it is of interest to note that hMGF-I promotescolony formation in this study. This observation is consistent withresults reported by other investigators where HIGF-I stimulated theproliferation of tumor cells (12).

Example 5 Effect of Ea-4-Peptide Gene on Colony Formation Activity

Overview: The effect of Ea-4-peptide gene on the colony formationactivity of MDA-MB-231 cells was adjudged

Methodology: MDA-MB-231 breast tumor cells were transfected with a geneconstruct containing an Ea-4-peptide gene and a green fluorescence(EGFP) gene and permanent transformants isolated. Both Ea-4-peptidetransformants and control cells are subjected to colony formation assayon a soft agar medium and colonies scored. FIG. 5 a illustrate theprocedure undertaken to transfect the gene construct and the methodutilized to determine colony formation of the transfected Ea-4 positiveclones as compared to EGFP controls.

Results: FIG. 5 b comprises photomicrographs of MDA-MB-231 breast tumorcells transfected with EGFP alone, and EGFP and Ea-4, with a graphinterposed there between that indicates the relative colony formationunder each scenario. While numerous colonies are observed in EGFPcontrol cells, no colony was observed in Ea-4-peptide transformed cells.

Example 6 The Effect of Ea-4-Peptide on Tumor Growth in Nude Mice

Overview: The effect of Ea-4-peptide gene on the tumor growth in nudemice was adjudged

Methodology: About 1.3×10⁷ ZR75-1 tumor cells, treated with troutEa-4-peptide (70 μg) were injected into the back of nude mice (threesites per animal). Two types of controls were used: (i) mice injectedwith 1.3×10⁷ ZR75-1 tumor cells treated with saline and (ii) miceinjected with protein preparation that does not contain Ea4-peptide.Eighteen days after tumor cell injection, the size of tumors developedin each mouse was measured. All mice were sacrificed at day 51 andtumors excised for weight measurement.

Results: FIG. 6 is a graph illustrating the effect of Ea-4-peptide ontumor growth. The results clearly show that the tumors developed fromZR75-1 cells treated with trout Ea4-peptide were only half in size andweight when compared with tumors developed from control mice. Therefore,trout Ea-4-peptide can inhibit the growth of tumors.

Example 7 The Effect of Ea-4-Peptide on Metastatic Cancer Invasiveness

Overview: Another characteristic of cancer cells is their ability toinvade normal tissues (metastasis) by migrating to other locations andcolonization. The molecular events of metastasis have become more clearin recent years. These events involve the secretion of metaloproteasesby tumor cells, digestion of basement membrane (invasion), and migrationand colonization of cancer cells in new locations (13). This invasivebehavior can be demonstrated by an in vitro invasion assay where themigration of cancer cells across the semi-solid Matrigel (proteinsisolated from basement membranes) is measured. To investigate whetherthe Ea-4-peptide of trout pro-IGF-I can retard the invasive activity ofcancer cells, an in vitro invasion assay was conducted in HT1080 cells,a known invasive cancer cell line, in the presence of Ea-4-peptide.

Methodology: Invasion assays were conducted in BIOCOAT MATRIGEL invasionchambers following the procedure provided by Becton Dickinson Labware(Bedford, Mass.; 40480 and 40481 guidelines). 1×10⁶ of HT1080 cells (aknown metastatic cancer cell line) in DMEM supplemented with 1.25% FBS,with or without various concentrations (4.2 and 8.4 nM) of the Ea-4peptide, were plated in each insert of the Matrigel or control invasionchambers. The inserts were placed in the respective chambers containingDMEM medium supplemented with 10% FBS, and entire chambers wereincubated at 37° C. under a humidified atmosphere of 5% CO₂ for 24 h.After removal of the non-invaded cells with cotton swabs, the invadedcells on the other side of the membranes were stained with theDiff-Quick™ stain (Becton Dickinson Labware, Bedford, Mass.) andobserved under an Olympic inverted microscope (magnification,200.times.).

Results: FIG. 7 is a table illustrating the effect of Ea-4-peptide onthe invasion activity of HT-1080 cells on Matrigell. As shown in thetable at FIG. 7, treatment of HT-1080 cells with Ea-4-peptide results ina dose-dependent reduction in the invasive activity of HT1080 cells. Theaddition of Ea-4 peptide to HT-1088 cells on Matrigell decreased theinvasive activity of HT-1080 cells from 60% to 14%, a 4-fold reductionof the invasive activity. Ea-4-peptide can therefore stop the metastasisof tumor cells during the expansion of tumor development.

Example 8 Effect of Ea-4-Peptide on Attachment of Cells

Overview: To determine whether treatment of transformed cells with theEa-4-peptide results in increased attachment of the cells to the culturedish.

Methodology: In the absence of fetal bovine serum, 293GP cells werecultured in a serum-free F12/DMEM supplemented with hIGF-I (2.6 nM) orEa-4-peptide (2.1 nM), respectively. After one week, the culture mediumwas removed, and cells were rinsed twice with PBS, fresh PBS was added,and the culture plates were shaken 20 times manually. Extent ofattachment was then determined.

Results: At the end of shaking, cells cultured in serum-free medium orserum-free medium supplemented with hlGF-I detached completely from theculture chamber while cells cultured in the serum free mediumsupplemented with Ea-4-peptide remained attached to the culture chamber.These results indicate that the Ea-4-peptide enhances the attachment ofoncogenic transformed cells to the culture chamber, similar to thebehavior exhibited by untransformed cells.

While the invention has been described with respect to preferredembodiments, those skilled in the art will readily appreciate thatvarious changes and/or modifications can be made to the inventionwithout departing from the spirit or scope of the invention as definedby the appended claims. All documents cited herein are incorporated intheir entirety.

1. A method of inhibiting the proliferation of a malignant cell,comprising administering to the malignant cell in vitro a nucleic acidcontaining a gene encoding a recombinant trout Insulin-like GrowthFactor (IGF-1) Ea2 or Ea4 peptide.
 2. The method of claim 1, wherein thetrout IGF-1 Ea2 or Ea4 peptide is selected from the group consisting of:a trout IGF-I Ea2 or Ea4 peptide homolog, and a trout IGF-I Ea2 orEa4-peptide fusion protein.
 3. The method of claim 2, wherein the troutIGF-1 Ea2 or Ea4E-peptide is from a rainbow trout.
 4. The method ofclaim 2, wherein the trout IGF-1 Ea2 or Ea4-peptide is selected from thegroup consisting of: Ea-2 domain peptide of SEQ ID NO:2, and Ea-4 domainpeptide of SEQ ID NO:4.
 5. The method of claim 4, wherein the nucleicacid encoding the trout IGF-1 Ea2 or Ea4-peptide is administered in apharmaceutical composition.
 6. A method for reducing the invasiveness ofmalignant cells, comprising administering to the malignant cells invitro a nucleic acid containing a gene encoding a recombinant troutIGF-I Ea2 or Ea4 peptide.
 7. The method of claim 6, wherein the troutIGF-1 Ea2 or Ea4-peptide is selected from the group consisting of: atrout IGF-1 Ea2 or Ea4-domain peptide homolog, and a trout IGF-1 Ea2 orEa4-domain peptide fusion protein.
 8. The method of claim 7, wherein thetrout IGF-1 Ea2 or Ea4-peptide is an E-domain peptide of a rainbowtrout.
 9. The method of claim 8, wherein the trout IGF-1 Ea2 orEa4-peptide is selected from the group consisting of: Ea-2 domainpeptide of SEQ ID NO:2, and Ea-4 domain peptide of SEQ ID NO:4.
 10. Themethod of claim 9, wherein the nucleic acid encoding the trout IGF-1 Ea2or Ea4-peptide is administered in a pharmaceutical composition.